GANP suppresses DNA recombination, measured by direct‐repeat β‐galactosidase gene construct, but does not suppress the type of recombination applying to immunoglobulin genes in mammalian cells

Abstract

Immunoglobulin V-region somatic hypermutation and C-region class-switch recombination are initiated by activation-induced cytidine deaminase (AID) in B-cells. AID-induced DNA damage at the immunoglobulin S-region is known to be repaired by non-homologous end-joining, but repair mechanisms at the V-region remain to be elucidated. In Saccharomyces cerevisiae , DNA homologous recombination is regulated by the expression of Sac3, involved in actin assembly, cell cycle transition and mRNA metabolism. Here, we demonstrate that the Sac3-homologue GANP suppresses DNA recombination in a direct-repeat β β β β -galactosidase gene construct in mammalian cells. Homozygous ganp gene knockout is embryonic lethal in mice. Embryonic fibroblasts immortalized from hetero-deficient ganp +/-mice showed more DNA recombination than wild-type. In contrast, over-expression of GANP suppressed either spontaneous DNA recombination or that caused by the introduction of aid cDNA into NIH3T3 cells (susceptible to I-sce I restriction enzyme cleavage but not to RAG-mediated immunoglobulin gene recombination). GANP suppresses the DNA recombination not only on the extrachromosomal DNA construct but also on the integrated DNA. The Sac3-homology portion is necessary for the suppressive activity, but the truncated carboxyl terminal MCM3-binding/acetylating region adversely augmented DNA recombination, acting as a dominant negative form. Expression of full-length GANP is critical for suppression of DNA hyper-recombination in mammalian cells. IntroductionB-cells produced in the bone marrow undergo random rearrangements of immunoglobulin ( Ig ) gene segments such as V H -D H -J H and V L -J L to generate unique antigen (Ag) receptors. Naïve B-cells recruited into peripheral lymphoid organs encounter exogenous Ags and start to proliferate and differentiate with the help of Th cells in the lymphoid follicles in response to T-cell-dependent Ag. The Ag-driven B-cells undergo Ig gene V-region somatic hypermutation and class-switch recombination initiated by activation-induced cytidine deaminase (AID), generating DNA cleavages during transcription or DNA replication in germinal centers (GCs) (Honjo et al . 2002). DNA double-strand breaks (DSBs) occur in both Ig Vregions and S-regions , which are considered to be repaired by the different DNA repair mechanisms. In contrast to 1206the S-region, where DNA repair is undertaken by a nonhomologous end-joining repair (NHEJR) mechanism utilizing Ku70/Ku80, DNA-PKcs, XRCC4 and DNA ligase IV, DSB repair mechanisms at the V-region genes remained undetermined (Casellas et al . 1998;Manis et al . 1998;Meek et al . 2004;Rooney et al . 2004). DNA repair in many cells depends on homologous recombination (HR) creating single-strand DNA to anneal with the undamaged complementary strand of the paired allele during DNA replication (West 2003). HR rescues the DNA with various DNA polymerases by reading the complementary strand from the undamaged allele.In Saccharomyces cerevisiae, an inhibitor of actin assembly, Sac3, is critic...

Lyn Signaling To Upregulate GANP Is Critical for the Survival of High-Affinity B Cells in Germinal Centers of Lymphoid Organs

“…GANP is involved in mRNA export in mammalian cells (30) and particularly in the selective export of Shugoshin-1 mRNA required for cell cycle progression through the M phase (20). The lack of GANP results in embryonic lethality at embryonic day 12.5 in mice and induces homology-mediated DNA hyperrecombination in cells, similar to that observed in suppressor of actin 3-deficient yeast cells (31,32). Alterations in GANP expression regulate the dsDNA breaks (DSB) at the IgV H region in GC B cells (33), suggesting involvement of GANP in the generation of DSBs or in DNA repair after translocation of the GANP/ AID complex to the target DNA in B cells.…”

GANP enhances the efficiency of mRNA nuclear export in mammalian cells

“…29 GANP has also been reported to suppress DNA recombination. 30 However, these observations do not explain why GANP is expressed in essentially all mammalian cells. Furthermore, because GANP also contains a region identical to MCM3AP (MCM3 acetylating protein), that interacts with and weakly acetylates MCM3, 31,32 these proteins have often been confused in databases and the literature.…”

“…Thus, recent work 34 has established a role for GANP distinct from those proposed previously, [27][28][29][30]33,47 which focussed on cells of the immune system in which GANP was discovered. Importantly, GANP is upregulated in a variety of lymphomas 28 and so the newly identified association of GANP with mRNA export in mammalian cells has broad implications for understanding its possible role in cancer progression.…”

Cholangiocarcinomas associated with long-term inflammation express the activation-induced cytidine deaminase and germinal center-associated nuclear protein involved in immunoglobulin V-region diversification

“…The mechanism of DNA hyper-recombination triggered by the lack of SAC3 has not yet been elucidated, but it is thought that the lack of SAC3, causing impaired mRNA export from the nucleus as the hrRNP complex, generated DNA injuries (24). The decreased expression of mammalian GANP also caused homology-mediated DNA hyper-recombination generated by the ectopic expression of AID in NIH-3T3 cells (15), suggesting that GANP coordinately functions in the regulation of DNA injuries caused by AID.…”

“…The middle portion of GANP is homologous to Saccharomyces cerevisiae SAC3, which is involved in mRNA export from the nucleus to the cytoplasm. SAC3-deficient cells demonstrate DNA hyper-recombination, as measured by using a tandem-repeat leu2 reporter gene (14); and similar activity has been demonstrated in GANP-deficient mouse cells (15). GANP is suggested to be involved in generation of SHM or modifying/repairing DNA injuries caused by cytidine deamination.…”